Technical Description: ErbB2 (Her2/Neu) overexpression is found in 20-30% of breast cancer patients as well as other cancers, and signifies poor prognosis. Success with humanized anti-ErbB2 antibodies has further validated ErbB2 as a therapeutic target. Ansamycin HSP90 inhibitors, such as 17AAG, are in phase l/ll clinical studies as novel anticancer agents as they target ErbB2 and other signaling proteins for ubiquitin-dependent degradation. Based on recent studies by us and others, we hypothesize that CHIP (C-terminus of HSP70-lnteracting Protein) and additional hitherto unknown ubiquitin ligases mediate 17AAG-induced ErbB2 downregulation either through proteasomal or through lysosomal degradation. Here, we propose comprehensive strategies to test these hypotheses. We will carry out cell biological, molecular and biochemical studies to distinguish whether HSP90 inhibitor-driven ErbB2 downregulation is mediated through degradation in the proteasome, lysosome or both. We will examine the role of CHIP and several candidate proteins identified through bioinformatics in 17AAG-induced ErbB2 ubiquitinylation and degradation, proliferation and survival of ErbB2 overexpressing breast cancer cell lines and in their in vivo growth in nude mice, using overexpression, dominant-negative mutant expression and RNAi knockdown strategies. If additional unknown ubiquitin ligases appear likely to mediate 17AAG-induced ErbB2 degradation, we will employ proteomics and yeast two-hybrid approaches to identify these proteins and characterize them functionally as for CHIP and other candidates. Through this comprehensive approach, we hope to elucidate the biological basis of ErbB2 downregulation by HSP90 inhibitory drugs and their anticancer activity. Success of these studies will open new therapeutic avenues for ErbB2-driven cancers as well those caused by EGFRvlll, an ErbB1 variant with biological similarities with ErbB2. Relevance to Public Health: This proposal will investigate new means of interfering with a cancer-causing protein Her2/Neu that is highly increased in 20-30% breast cancers with worst prognosis. The proposed studies could provide a basis for newer, more effective targeted therapies against breast and other cancers.